Highly Efficient Synthesis of Rare Sugars from Glycerol in Endotoxin-Free ClearColi by Fermentation

Abstract

Rare sugars possess potential applications as low-calorie sweeteners, especially for anti-obesity and anti-diabetes. In this study, a fermentation biosystem based on the "DHAP-dependent aldolases strategy" was established for D-allulose and D-sorbose production from glycerol in endotoxin-free ClearColi BL21 (DE3). Several engineering strategies were adopted to enhance rare sugar production. Firstly, the combination of different plasmids for aldO, rhaD, and yqaB expression was optimized. Then, the artificially constructed ribosomal binding site (RBS) libraries of aldO, rhaD, and yqaB genes were assembled individually and combinatorially. In addition, a peroxidase was overexpressed to eliminate the damage or toxicity from hydrogen peroxide generated by alditol oxidase (AldO). Finally, stepwise improvements in rare sugar synthesis were elevated to 15.01 g/L with a high yield of 0.75 g/g glycerol in a 3 L fermenter. This research enables the effective production of rare sugars from raw glycerol in high yields.

Keywords: ClearColi; D-allulose; aldolase; glycerol; rare sugars.

Figure 1

Construction of synthetic pathway for rare sugar production from glycerol in ClearColi.

Figure 2

Effects of different overexpression manners for related enzymes on the production of rare sugars. (A) Schematic diagram of different plasmid combinations. (B) Production of rare sugars using different plasmid combinations.

Figure 3

Evaluation of the strength variations of GFP after modulating the gfp gene with the RBS library.

Figure 4

Rare sugar titer detection after modulating aldO, rhaD, and yqaB genes individually with the RBS library. (A) Modulating aldO with the RBS library. (B) Modulating rhaD with the RBS library. (C) Modulating yqaB with the RBS library. The blue dashed line represents the relative titer of rare sugars for the control without RBS modulation.

Figure 5

Rare sugar titer detection after dual modulating aldO and rhaD with RBS libraries. The blue dashed line represents the relative titer of rare sugars for the control (strain Y21).

Figure 6

Quantification of rare sugar production for the representative strains after modulating with RBS libraries.

Figure 7

Effect of peroxidase overexpression on the production of rare sugars. (A) Effects of peroxidase overexpression on the growth of recombinant strains. (B) Production of rare sugars using the recombinant strains overexpressing peroxidases.

Figure 8

Time profiles of rare sugars and residual glycerol in the engineered strain. The red arrow was the point of supplementation with 10 g/L of glycerol.